Statine-based peptidomimetic compounds as inhibitors for SARS-CoV-2 main protease (SARS-CoV‑2 Mpro)

COVID-19 is a multisystemic disease caused by the SARS-CoV-2 airborne virus, a member of the Coronaviridae family. It has a positive sense single-stranded RNA genome and encodes two non-structural proteins through viral cysteine-proteases processing. Blocking this step is crucial to control virus replication. In this work, we reported the synthesis of 23 statine-based peptidomimetics to determine their ability to inhibit the main protease (Mpro) activity of SARS-CoV-2. Among the 23 peptidomimetics, 15 compounds effectively inhibited Mpro activity by 50% or more, while three compounds (7d, 8e, and 9g) exhibited maximum inhibition above 70% and IC50 < 1 µM. Compounds 7d, 8e, and 9g inhibited roughly 80% of SARS-CoV-2 replication and proved no cytotoxicity. Molecular docking simulations show putative hydrogen bond and hydrophobic interactions between specific amino acids and these inhibitors. Molecular dynamics simulations further confirmed the stability and persisting interactions in Mpro's subsites, exhibiting favorable free energy binding (ΔGbind) values. These findings suggest the statine-based peptidomimetics as potential therapeutic agents against SARS-CoV-2 by targeting Mpro.


General information
Reagents were purchased from Sigma-Aldrich Co.All solvents were purchased as reagent grade, dried using standard conditions, and stored over molecular sieves.Purification of products was carried out using silica gel flash chromatography (Whatman 60, 230-400 mesh).NMR analyses were performed on a Varian Unity Plus-300 spectrometer.Melting points were obtained on a Thomas Hoover capillary melting point apparatus and are uncorrected.All compounds are > 95% pure by high-resolution mass spectra (HRMS) that were performed on a Waters Micromass Q-Tof Micromass spectrometer equipped with a lock spray source.The IR spectra were obtained on a Perkin-Elmer spectrometer model Spectrum One in liquid film or KBr pellets.Optical rotation measurements were determined on a Perkin-Elmer 341 LC polarimeter.

General procedure for compounds 7a-h
To a 0 ºC cooled mixture of compound 1 (0.2 g; 0.647 mmol) and the appropriate methyl ester hydrochloride (1.15 mmol) in dry CH 2 Cl 2 (10 mL) were added EDC.HCl (0.186 g; 0.970 mmol), HOBt (0.13 g; 0.970 mmol) and N-methylmorpholine (0.21 mL; 1.94 mmol).The mixture was stirred at room temperature for 24 h, and the volatiles were removed under reduced pressure.The resulting residue was dissolved in CH 2 Cl 2 (50 mL) and successively washed with 5% H 3 PO 4 (50 mL), 20% Na 2 CO 3 (50 mL), water (40 mL), and brine (50 mL) and dried with Na 2 SO 4 after which it was filtered and evaporated under reduced pressure.The products were purified by flash chromatography on silica gel using EtOAc/hexane as eluents.

General procedure for compounds 8a-g
The corresponding ester 7a-g (5.0 mmol; 1 eq.) was solubilized in distilled dichloromethane (5.0 mL).Anhydrous pyridine (7.5 mmol; 0.6 mL; 1.5 eq.) and DMAP (5.0 mmol; 1 eq.) were added to the solution.The mixture was cooled to 0 °C and then acetic anhydride (7.5 mmol; 1.5 eq.) was added dropwise.The reaction was stirred at room temperature for 3 h until complete consumption of the starting material.The reaction was diluted with dichloromethane (50 mL) and extracted successively with water (50 mL) and brine (50 mL).The organic phase was dried over anhydrous sodium sulfate and then evaporated on a rotary evaporator.Purification by chromatographic column on silica gel afforded the products as solids.

General procedure for compounds 9a-g
The corresponding ester (7a-h) (5.0 mmol) was solubilized in a minimum volume of distilled dichloromethane.The resulting solution was cooled to 0 °C and then trifluoroacetic acid (7.50 mmol, 0.57 mL, 1.5 eq) was added dropwise.The reaction was stirred at room temperature for 3 h until complete consumption of the starting material and then completely evaporated in a rotary evaporator.The resulting product was purified by recrystallization from diethyl ether.

In vitro inhibition of SARS-CoV-2 Mpro
Recombinant SARS-CoV-2 Mpro synthetic gene expressed in E. coli BL21(DE3)pLysS cells were used in a fluorescent resonance energy transfer (FRET) assay, using as substrate the DABCYL-AVLQ↓SGFRLL-EDANS peptide (Biomatik Corp., CA), and as positive control a synthetic dipeptide covalent inhibitor of SARS-CoV's Mpro, GC-376 (PubChem CID 71,481,119).The enzyme concentration was fixed at 1.5 μM, the substrate at 50 μM and the compounds (statine-based peptidomimetics and GC-376) ranged from 0.001 to 1000 μM.The enzyme and compounds were incubated in 5 mM NaCl, 20 mM Tris.HCl pH 8.0, 5 mM DTT for 15 min at 37ºC before starting with the substrate.The emission fluorescence of EDANS was monitored in the following parameters: λ exc = 330 nm, λ em = 490 nm, at 37 °C for 45 min.Fluorescence data (RFU) was converted into substrate cleavagespecific activity using fluorescent conversion factor (FEC) previously calculated based on the EDANS-DABCYL fluorophore pair.Maximum enzyme activity was considered in the situation with vehicle (DMSO), and the values were used to calculate the enzyme inhibition by the compounds.The concentration that inhibits 50% of the enzyme activity (i.e., the half-maximal inhibitory concentration, IC 50 ) was calculated in the software GraphPad Prism 9.0.

Antiviral and cytotoxicity assays
We state that human participants are not involved in the study.We evaluated the compounds' biological activity in a cell model using Calu-3 cells, a human submucosal gland cell line.Calu-3 cell models are widely used as a preclinical model for respiratory disease drug screening due to their bronchiolar epithelium characteristics and ability to replicate viruses with higher titles, including SARS-CoV-2 [19][20][21][22] .
The cytotoxicity assay consisted of interaction between the compounds 7d, 8e, and 9g, at different concentrations (200, 100, 50, 25, and 12.5 µM) with Calu-3 cells (kindly donated by the Farmanguinhos platform RPT11M) at a cell density of 1 × 10 4 cells/well for 72 h.Afterward, the cells were submitted to viability evaluation by methylene blue assay.For this assay, cells were washed with PBS 1 × and stained with methylene blue solution (Hanks' solution (HBSS), 1.25% glutaraldehyde, and 0.6% methylene blue) for 1 h.Then, the cells were rewashed and elution solution (50% ethanol, 49% PBS 1x, and 1% acetic acid) was added for 15 min.After that time, the absorbance was read at 660 nm in the spectrophotometer.
All the compounds used in vitro assays were resuspended in 100% DMSO (dimethyl sulfoxide), aliquoted, and stored at − 20 °C to avoid compound degradation 23 .The DMSO final concentrations for each molecule's test concentration were equal or lower than 1% (v/v) diluted in DMEM (Dulbecco's Modified Eagle Medium) not affecting the growth of the cells 24,25 .According to WHO guidelines 26 , all virus manipulation was realized at a biosafety level 3 (BSL3) multiuser facility.

Statistical analysis
The graphs were created using the GraphPad Prism 9.0 software and represent the middle of the results for each experiment realized with a minimum of three technical replicates.We determined the EC 50 and CC 50 values by Nonlinear regression of Log(inhibitor) or inhibitor vs. Normalized response of best curve generated (R 2 values ≥ 0.9).

Protein and ligand structures preparation
The molecular docking simulations were performed with the crystallographic structure of the SARS-CoV-2 Mpro enzyme obtained in a covalent complex with an irreversible peptide-like inhibitor named , available in the Protein Data Bank as PDB ID: 6LU7 (resolution = 2.16 Å) 27 .The missing residues were added using the CHARMM-GUI platform (http:// www.charmm-gui.org/) 28 , defining the protonation state in physiological pH 7.4, which was predicted by pdb2pqr server (https:// server.poiss onbol tzmann.org/ pdb2p qr) and removing water molecules.The three-dimensional structures of the statine-like derivatives 7d, 8e, and 9g were drawn using ChemDraw v. 20.0 29 considering their protonation state in physiological pH 7.4, and geometry optimization was performed using the MMFF94 force field available in the Spartan (v.10) software (Wavefunction, Inc. https:// www.wavef un.com).Then the structures were converted to the pdbqt format using the Open Babel chemical toolbox 30 .

Molecular docking
The molecular docking simulations were performed using the AutoDock Vina 1.1.2program 31 and prepared in the AutoDockTools (ADT) (v.1.5.6) 32 graphical interface according to the protocol and parameters previously described by our research group 33 , considering the physiological pH 7.4.The docking protocol was validated by redocking the N3 inhibitor as a non-covalent ligand.The N3 inhibitor was removed from the structure, and the binding orders were restored for the inhibitor interacting with Cys145 amino acid.The root-mean-square deviations (RMSD) calculations of the 10 pose results were carried out using the PyMOL (v.3.5) software 34 , considering the best results RMSD < 2.0 Å. Ligand-Mpro complexes were analyzed for the main intermolecular interactions, such as hydrogen bond (H-bond) and hydrophobic interactions, with the PyMOL, and the images of the binding poses were composed with the Visual Molecular Dynamics (VMD) (v.1.9.4) software 35 .
Vol:.( 1234567890 www.nature.com/scientificreports/Molecular dynamics Molecular dynamics simulations were carried out in triplicate with the GROMACS 2022 package 36 using the CHARMM36 force field 37 with the top-ranked pose of each ligand-Mpro complex obtained by molecular docking applying the protocol described previously by our research group 33 .The ionization states of the protein's residues were adjusted to pH 7.4 using the pdb2gmx Python script.Ligand-protein complex was included in a periodic triclinic box (box dimensions: 5.416 × 4.538 × 4.348 nm and box volume: 854.88 nm 3 ), solvated with the TIP3P model of water, and neutralized with 8 atoms of Na + ions to neutral ligands (7d and 8e) and 7 Na + atoms to charged ligand (9g).RMSD, RMSF, hydrogen bonding, and cluster analysis were made using gmx rms, gmx rmsf, gmx hbond, and gmx cluster modules available in the GROMACS package.The ΔG bind was calculated by the MM-PBSA method, applying g_MMPBSA module v.5.1.2 38considering the internal dielectric of the protein solute of 2. The energy contribution of residues was calculated with MmPbSaStat.py and MmPbSaDecomp.pyscripts 38 .H-bonding frequencies were calculated with HbMap2Grace 39 software.Figures of the interactions and trajectories analysis were composed with the VMD 35 software.
The acetylation reactions were carried out using acetic anhydride and DMAP in a basic medium affording the final compounds 8a-g 45 .The N-Boc deprotection of 7a-h compounds was made using trifluoroacetic acid generating peptidomimetics 9a-g 46 .Finally, the hydrazide compound 10 was obtained from the corresponding ester 7 h (R = -CH 2 Ph) and hydrazine hydrate in methanol (Fig. 2).

Inhibition of SARS-CoV-2 Mpro
The ability of compounds to inhibit the SARS-CoV-2 Mpro activity was assessed by an in vitro FRET-based assay.Table 2 shows % of maximum inhibition (efficacy) compared to negative control (vehicle, DMSO) and IC 50 (potency) values.The most promising compounds were selected based on efficacy (high maximum inhibition values) and potency (low half maximum inhibition, IC 50 values).As a positive control, we used the GC-376 (PubChem CID 71,481,119) compound, a small synthetic dipeptide previously identified as a covalent inhibitor of SARS-CoV's Mpro and used in several studies as gold standard for in vitro Mpro inhibition assay 47,48 .In our system, the maximum inhibition achieved by GC-376 was 75%, presenting an IC 50 = 0.541 µM (Table 2).Sixteen out of 19 tested compounds inhibited Mpro activity by 50% or more (Table 2) and maximum inhibition was achieved with compound 9g, which inhibited the enzyme activity by 80% compared to negative control (vehicle).
Only five compounds (7d, 8c, 8d, 8e, and 9g) were able to inhibit Mpro activity by 60% or more at low IC 50 values (Table 2), while all the other compounds only exerted inhibitory effects (≥ 50%) at high IC 50 values (IC 50 > 770 μM).Therefore, we selected the three most promising compounds (7d, 8e, and 9g), which showed 70% or more inhibition than standard GC-376 and had an IC 50 of less than 1 µM (Table 2).These compounds were further evaluated for their ability to inhibit virus replication.www.nature.com/scientificreports/

In silico molecular docking and dynamics simulations
The potential binding mode and main intermolecular interactions of the statine-based derivatives 7d, 8e, and 9g into the active site of the SARS-CoV-2 Mpro were evaluated through molecular docking following the protocol previously reported by our research group 33 .The docking protocol used in our study was validated by redocking, considering the N3 inhibitor as a non-covalent inhibitor.The bond between N3 and the enzyme was broken, and its double bond was restored.Our analysis considered 14 rotatable bonds, as the N3 has 4 amide bonds.
It is worth mentioning that the Mpro (PDB ID: 6LU7) co-crystalized inhibitor, N3, is an irreversible peptidelike inhibitor 27 .According to its redocking pose, N3 shows similar H-bonding interactions with residues of subsites S1 (Phe140), S2 (His41 and Glu166), and S4 (Gln189) (Figure S1), as seen in the statine-like derivatives proposed as non-covalent inhibitors, that shared similar binding mode, at least with two subsites as this inhibitor.
The molecular dynamics simulations (MD) were carried out in triplicate, starting with the top-ranked poses of 7d, 8e, and 9g with SARS-CoV-2 Mpro (PDB ID: 6LU7), were performed to evaluate the behavior of these ligand-protein complexes in an aqueous system, during 200 ns, using the GROMACS software 55 with Charmm36 force field 37 .
In the first instance, the compounds were docked into the Mpro's active site, and as mentioned before, they remained close to the Cys145-His41 catalytic dyad region.To confirm if this specific area would encourage favorable and persistent interactions, we conducted an RMSD (root-mean-square deviations) analysis of the ligands over a 200 ns simulation period.
The RMSD analysis for compound 7d showed a tendency to leave the active site after 70 ns of simulation, presenting an RMSD value of 11.6 ± 6.40 Å and a high standard deviation (Fig. 5a).Compound 8e presented relative stability and persistence into the active site at the beginning of the simulation (0-40 ns); after that, left the active site presenting an RMSD value of 26.3 ± 7.34 Å (Fig. 5b).The derivative 9g showed RMSD = 34.7 ± 12.8 Å, with low persistence in the active site (about 15 ns).Based on the 200 ns MDS analysis, it can be stated that inhibitor 7d has the strongest interaction with the protein's active site.While the other inhibitors may exhibit good inhibition values, they cannot remain in contact with this region for an extended period.
It is interesting to note that the root-mean-square-fluctuation (RMSF) calculation for 7d indicates that, even when this ligand is inside and out of the binding site cavity, the residues have mobility greater than 2.0 Å for Cα atoms of Asp187(S2), Gln189(S4), and Thr190(S5) (Fig. 7a).Considering the movement 8e during the 200 ns of simulation, we evaluated the difference in the RMSF of the Cα atoms in two time intervals: 1-40 ns and 40-200 ns (Fig. 7b).In general, the ligand induces a gain of 0.98 to 2.10 Å in the mobility of residues belonging Regarding the statine-like derivative 9g, the RMSF values were analyzed considering two-time intervals (1-15 ns, 15-200 ns) that indicated fluctuations above 2 Å for residues belonging to subsites S2 (His41, Tyr54, and Met49) and S5 (Thr190) predominantly (Fig. 7c).
The analysis of intermolecular interactions via hydrogen bonding of ligand 7d exhibits a hydrogen bonding interaction with the residue Glu166(S1), His163(S1), and Ser144, with a great lifetime observed between 18 and 30%.In addition to H-bond interactions with catalytic residues Cys145 and His41 with low persistence, 5.19 and 8.97 respectively (Table 4).
The analysis of the 8e-Mpro complex revealed those with the longest duration involving the ligand and the following residues: Glu166(S1) (43.8%) and Gln189(S4) with short lifetime from 6.26 to 15% (Table 4).Furthermore, FDA-approved Paxlovid™ (nirmatrelvir + ritonavir) was the first oral antiviral for mild to moderate COVID-19 cases in adults on May 25, 2023 59 .Nirmatrelvir inhibits viral replication by bonding to Cys145 catalytic residue from Mpro and forming hydrogen bonds with catalytic His164, Glu166(S1), and Gln189(S4) 60 .This finding supported the acetylated statine-like derivative 8e, which demonstrates the potential of binding www.nature.com/scientificreports/resulting in the highest energy cost of desolvation of the binding site (44.5 kcal/mol) when compared to the other compounds (~ 20 kcal/mol) (Table 5).It was noted that ΔG bind energy analysis of the ligands before moving out from the binding site was consistent, as previously discussed.Although derivative 9g remained in the active site for less time than 8e and 7d, this interaction was sufficient to cause inhibition of the enzyme, which resulted in its best binding free energy value of − 57.7 kcal/mol observed (Table 5).

Conclusions
Targeting the SARS-CoV-2 main protease (Mpro), 23 statine-based peptidomimetics were synthesized and tested for their ability to inhibit the Mpro activity.The three most effective compounds (7d, 8e, and 9g) could inhibit the Mpro enzyme activity in the sub-micromolar range.These compounds have been found to be non-cytotoxic and can suppress about 80% of the replication of the SARS-CoV-2 virus.In silico studies have also shown that these compounds are stable and have persistent interactions with the Mpro active site, indicating their potential as inhibitors.By blocking the activity of the main protease, which is essential for viral replication, these compounds have the potential to inhibit virus replication with low micromolar EC 50 .Finally, we found new hit compounds that could lead to promising drug candidates against the COVID-19 disease.Table 5.The binding free energy (ΔG bind ) terms of the ligand-Mpro complexes calculated for 7d, 8e, and 9 g with the MM-PBSA method (mean ± standard deviation energies; kcal/mol): van der Waals (ΔE vdW ), electrostatic (ΔE elect ), solvation (ΔE solv ), and solvent accessible surface area (ΔE sasa ).

Figure 4 .
Figure 4. Best pose by molecular docking simulations of statine-like derivatives on the SARS-CoV-2 Mpro active site (PDB ID: 6LU7): (A) 7d; (B) 8e, and (C) 9g.The residues involved in H-bond interactions (dashed black lines) with the ligands are in ball-and-line model (cyan color) and residues involved in hydrophobic interactions are in stick models (light green color).In 2D structures, the atoms (or groups) of the ligands involved in H-bond interactions are circled in yellow.

Table 2 .
Inhibition of SARS-CoV-2 Mpro proteolytic activity by the statine-based peptidomimetics.NT not tested, ND not determined.